Electron microscopic studies of carboxysomes of Thiobacillus neapolitanus

The outer part of the carboxysomes of Thiobacillus neapolitanus was examined by electron microscopy using negatively stained, cryo-treated, frozen hydrated and freeze dried specimens. From stereo-micrographs of freeze dried and fixated carboxysomes the three dimensional structure of the carboxysomes was elucidated. The carboxysomes always appear as hexagonal bodies, which possess twelve pentameric planes. This indicates that carboxysomes have the form of a pentagonal dodecahedron. Inside the carboxysomes the ribulose-1,5-bisphosphate carboxylase molecules are arranged in rows and concentric rings. Negatively stained and cryo-treated carboxysomes do not differ significantly in size. The mean size of these carboxysomes is 117.3±6.9 nm (n=782)     

Relations between d-ribulose-1,5-bisphosphate carboxylase,carboxysomes and CO2 fixing capacity in the obligate chemolithotroph Thiobacillus neapolitanus grown under different limitations in the chemostat

An adaptation of the d-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity to changing CO₂ concentrations in the growth medium in the chemostat was observed in the obligate chemolithotroph Thiobacillus neapolitanus. RuBPCase activity has been separated in a soluble and particulate fraction. The activity of the particulate fraction appeared to be associated with the carboxysomes.The total activity of RuBPCase of CO₂ limited cultures was about 5-fold higher than the activity of thiosulphate limited cultures grown in the presence of 5% CO₂ whilst the particulate activity and the soluble activity were about 8- and 1.5-fold higher, respectively. The fluctuation of the total and particulate RuBPCase activity correlated with the changes in volume density of carboxysomes in the cell.An inverse correlation between maximal CO₂ fixing capacity by whole cells and the volume density of carboxysomes was observed. The change in ratio of soluble RuBPCase activity to particulate RuBPCase activity paralleled the change in maximal CO₂ fixation by whole cells during the different growth conditions.     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Occurrence,structure and function of intracellular polyglucose in the obligate chemolithotroph Thiobacillus neapolitanus

Nitrogen-limited cells of the obligate chemolithotroph Thiobacillus neapolitanus formed an intracellular polymer during growth in the chemostat. This polymer was isolated and characterized as a branched polyglucose composed of units joined by -14 and -16 linkages. Polyglucose in T. neapolitanus can be considered a storage compound since formation of this compound took place during excess of energy and CO₂ whilst shortage of CO₂ resulted in rapid breakdown of polyglucose. Moreover the breakdown of polyglucose generated metabolically useful energy as could be demonstrated by polyglucose-dependent protein synthesis. Possession of polyglucose did not influence the viability of T. neapolitanus during prolonged periods of energy starvation. Activities of key enzymes of the oxidative pentose phosphate cycle, glucose-6-phosphatedehydrogenase and 6-phospho-gluconate-dehydrogenase, were demonstrated in cell free extracts of T. neapolitanus and appeared to increase 5- and 3-fold, respectively, during growth on NO ₃ ^- instead of NH ₄ ⁺ as a nitrogen source.     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

The specific activity of ribulose-1,5-bisphosphate carboxylase in relation to genotype in wheat

The specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) in crude extracts of leaves from euploid, amphiploid and alloplasmic lines of wheat fell into high or low categories (3.75 or 2.70 mol·mg^–1·min^–1, 30°C). For the alloplasmic lines, where the same hexaploid nuclear genome was substituted into different cytoplasms, the specific activity of RuBPCase was consistent with the type of cytoplasm (high for the B and S cytoplasms and low for the A and D cytoplasms). There was no evidence from the euploid and amphiploid lines that small subunits encoded in different nuclear genomes influenced the specific activity. High specific activity was conferred by possession of the chloroplast genome of the B-type cytoplasm which encodes the large subunit of RuBPCase. All lines with a cytoplasm derived from the Sitopsis section of wheat, with the exception of Aegilops longissima and A. speltoides 18940, had RuBPCase with high specific activity. In contrast with the euploid lines of A. longissima, the alloplasmic line containing A. longissima cytoplasm from a different source had RuBPCase with high specific activity. The difference in specific activity found here in-vitro was not apparent in-vivo when leaf gas exchange was measured.Abbreviation RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

Thermal properties and oxygenase activity of ribulose-1,5-bisphosphate carboxylase from the thermophilic purple bacterium, Chromatium tepidum

Abstract Ribulose-1,5-biphosphate carboxylase (RuBPCase) partially purified from the thermophilic purple bacterium Chromatium tepidum displayed maximum carboxylase activity at 50°C, while enzyme from a related mesophilic species, Chromatium vinosum , was completely inactive at 50°C. RuBPCase from C. tepidum showed ribulose-1,5- bisphosphate-dependent oxygenase activity, and, in addition, O₂ was found to partially destroy carboxylase activity. It is concluded that thermophilic purple bacteria produce heat-stable RuBPCase and that all RuBPCases, even those from an obligate anaerobe such as C. tepidum , have associated oxygenase activity.     

Purification and properties of ribulose 1,5-bisphosphate carboxylase from sunflower leaves

Extracts from sunflower leaves possess a high ribulose-1,5-bisphosphate (RuBP) carboxylase capacity but this enzyme activity is not stable. A purification procedure, developed with preservation of carboxylase activity by MgSO₄, yielded purified RuBP carboxylase with high specific activity (40 nkat mg^-1 protein). Measurement of kinetic parameters showed high Km values (RuBP, HCO ₃ ^- ) and high Vmax of the reaction catalyzed by this sunflower enzyme; the results are compared with those obtained for soybean carboxylase. Enzyme characteristics are discussed in relation to stabilization and activation procedures and to the high photosynthesis rates of this C₃ species.     

Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans Synechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the / barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in V _max but did not alter the enzyme's relative specificity towards either of its gaseous substrates, CO₂ and O₂. However replacement of alanine 340 with glutamate decreased the enzyme's specificity for CO₂ but had no significant effect on either the K _m for ribulose-1,5-bisphosphate or CO₂ or on V _max. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - CABP 2-carbox-yarabinitol-1,5-bisphosphate We thank Karen Moore for the statistical analysis of the specificity factors. We acknowledge helpful discussions with Jim Pitts and Richard Pickersgill. This work was aided by the invaluable technical assistance of Iain Major.     

Structural framework for catalysis and regulation in ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the enzyme assimilating CO2 in biology. Despite serious efforts, using many different methods, a detailed understanding of activity and regulation in Rubisco still eludes us. New results in X-ray crystallography may provide a structural framework on which to base experimental approaches for more detailed analyses of the function of Rubisco at the molecular level. This article gives a critical review of the field and summarizes recent results from structural studies of Rubisco.     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

Abstract The evolutionary relationship of the RuBisCO large subunit gene(s) ( rbcL ) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum , and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon , and Mastigocladus laminosus . In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus , and Thiothrix nivea . The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus , and T. nivea . The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus . It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].     

Cloning and expression of the d-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli

Abstract Both form I and II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were detected in Thiobacillus intermedius by heterologous hybridization using specific probes from Anacystis nidulans and Rhodobacter sphaeroides , respectively. However, only the previously reported from I enzyme could be demonstrated in cells grown under a number of different conditions. The reason(s) why the form II gene is not expressed in T. intermedius is/are not clear at this time. The form II gene was isolated from a lambda library by screening with the Rb. sphaeroides probe. A Sal I fragment from this clone was ligated into pUC8 and transformed into Escherichia coli DH5α. Subclones pTi20IIA and pTi20IIB representing both orientations relative to the lac promoter were isolated. Low levels of RuBisCO activity were detected in both induced and non-induced pTi20IIA indicating the probable expression from a T. intermedius promoter. Induced pTi20IIB produced much higher levels of enzyme activity. Analysis of cell-free extracts using sucrose density gradients confirmed the expression of a form II RuBisCO similar in size to that found in Rhodobacter capsulatus . Other Calvin cycle genes were not clustered with either the form I or form II genes.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

Activation of ribulose-1,5-bisphosphate carboxylase by chloroplast metabolites in a reconstituted spinach chloroplast system

In a preparation of soluble components from isolated spinach (Spinecia oleracea L.) chloroplasts, the activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) is strongly increased by 6-phosphogluconate or by NADPH at pH 8.0. When the thylakoid system is added to these soluble components (reconstituted chloroplast system) plus ferredoxin, the carboxylase is even more strongly activated in the light. This light activation appears to be due to reduction of endogenous NADP⁺ by electrons from the light reactions transferred via ferredoxin, since NADPH alone can activate the purified enzyme in the dark while reduced ferredoxin does not. The regulatory properties of the enzyme in the reconstituted chloroplast system are compared with those of the isolated enzyme, and their possible physiologic significance is discussed.Abbreviations Fd ferredoxin - PPC pentose phosphate cycle - 6-PGluA 6-phosphogluconate - Rib-5-P ribose-5-phosphate - RuBP ribulose-1,5-bisphosphate     

Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from the green alga Chlamydomonas reinhardtii. Incubation of either CO₂/Mg²⁺ -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO₂/O₂ specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The K _m values for RuBP were not significantly changed for the mutant enzymes, but the V _max values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO₂/O₂ specificity of Rubisco.Abbreviations and Symbols CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K _c K _m for CO₂ - K _o K _m for O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation Paper No. 9313, Journal Series, Nebraska Agricultural Research DivisionThis work was supported by National Science Foundation grant DMB-8703820. We thank Drs. Archie Portis and Raymond Chollet for their helpful comments, and also thank Dr. Chollet for graciously providing CABP and [¹⁴C]CABP.